The present invention relates to a method for preparing a transformant lacking a selective marker gene using site-specific recombination of yeast. After a gene of interest is transferred into yeast by using this method, a transformant of yeast deprived of a selective marker gene and transfected with a desired character can be obtained. Yeasts obtained by transformation methods of the present invention can be used to produce liquors or bread based on yeasts of the genus Saccharomyces, and are especially useful in producing beer.
Although many gene transfer methods concerning yeasts have been reported, all of these methods require a selective marker for selecting a transformant because of low gene transfer efficiency. Selective markers include those restoring auxotrophy, but typically consist of resistance genes to drugs such as antibiotics because auxotrophy is often difficult to confer to yeasts. However, it would be desirable to remove and reuse selective marker genes to repeatedly transform the same strain because few classes of drug resistance genes can be efficiently used in yeast. It would be also desirable to remove selective marker genes from transformants from the aspect of safety in commercialization of recombinants.
In order to solve these problems, methods for removing selective marker genes from transformants have been developed. An example of these methods uses site-specific recombination.
Site-specific recombination occurs when a recombinase acts on two recognition sequences consisting of specific nucleotide sequences recognized by said recombinase to induce recombination between said recognition sequences. These recombinations invite such events as deletion, insertion or inversion according to the arrangement of a pair of recognition sequences. Four site-specific recombinations are known, ie, Cre/lox derived from bacteriophage P1, FLP/FRT derived from Saccharomyces cerevisiae, R/RS derived from Zygosaccharomyces rouxii and Gin/gix derived from bacteriophage Mu (each designated by the combination of a recombinase and the specific nucleotide sequence recognized by the recombinase).
Saccharomyces cerevisiae is known to have a cyclic double-stranded DNA called the 2 xcexcm plasmid in the cells, and the presence of a site-specific recombination mechanism in the 2 xcexcm plasmid has been shown (Broach, J. R., Guarascio, V. R. and Jayaram, M., Cell, 29, 227-234, 1982). The 2 xcexcm plasmid is a cyclic plasmid of 6318 bp, which is known to have a pair of inverted repeats of 599 bp in its molecule and to undergo site-specific recombination between these inverted repeats. The recombination site between these inverted repeats contains a spacer sequence of 8 bp flanked by short inverted repeats of 13 bp containing one mismatch (FRT sequences) and followed by another 13-bp inverted repeat at one end. Site-specific recombination occurs when a recombinase (Flp protein) expressed by the FLP gene encoded by the plasmid itself acts on the FRT sequences consisting of a specific nucleotide sequence present in the recombination site in the inverted repeats.
A known FRT sequence is a 34-bp sequence consisting of a spacer sequence of 8 bp and inverted repeats of 13 bp (J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA, 82, 7270-7274, 1985). However, this 34-bp FRT sequence is not suitable for commercial use because site-specific recombination using this sequence leaves recognition sequences of the recombinase on chromosomes after recombination so that undesired recombination may be induced.
Thus, there was a need for suppressing recombination between recognition sequences left on chromosomes after recombination.
Some groups reported excision of selective marker genes using site-specific recombination with a recombinase and the recognition sequences of the recombinase, such as excision of selective marker genes using the FLP/FRT system in Saccharomyces cerevisiae (F. Storici, M. Coglievina and C. V. Bruschi, Yeast, 15, 271-283, 1999). Storici et al. used the kanMX4 gene or URA3K1 gene as a selective marker gene to transform Saccharomyces cerevisiae by integrating the selective marker gene between FRT sequences recurring in the same orientation in the case of Cir+ strains carrying the 2 xcexcm plasmid or integrating the selective marker gene together with the FLP gene between similar FRT sequences in the case of Cir0 strains lacking the 2 xcexcm plasmid. The resulting transformant was cultured in a non-selective medium to induce recombination between FRT sequences so that the selective marker gene was successfully removed. Taking advantage of the fact that a nucleotide change in the core (a 8-bp spacer sequence) of an FRT sequence induces recombination with a sequence having the same nucleotide change but suppresses recombination with a sequence having another nucleotide change, they suppressed undesired recombination between FRT a sequences left on chromosomes by using an FRT sequence having a different nucleotide change at each run of repeated transformation and excision of a selective marker gene. However, the selective marker gene excision efficiency of their method is as low as 0.01%-1.39% and thus it is not easy to select strains deprived of selective marker genes. Moreover, the number of nucleotide changes at the core of a FRT sequence is limited.
A method using the Zygosaccharomyces rouxii-derived site-specific recombination system R/RS in Saccharomyces cerevisiae was also developed (JPA 66587/98). According to this document, a selective marker gene and the R gene linked to a galactose-inducible promoter were integrated between RS sequences recurring in the same orientation to transform Saccharomyces cerevisiae. Several nucleotides were deleted from the outside of each of a pair of RS sequences flanking the R gene and the selective marker gene to suppress undesired recombination with RS sequences left after recombination. However, this method involved introducing a foreign gene for recombinase (R gene). Moreover, the excision efficiency of the selective marker gene varies with the strain of yeast to be transformed, and it is not easy to select strains deprived of a selective marker gene especially in commercial strains such as brewer""s yeasts and wild-type yeasts due to the low excision efficiency of the selective marker gene.
A sequence which inhibits growth of cells when it is highly expressed in Saccharomyces cerevisiae (growth inhibition sequence) has been reported. Excision of selective marker genes with such a sequence has already been reported (M. Kawahata et al., Yeast 15, 1-10, 1999). Kawahata et al. inserted the URA3 gene and a growth inhibition sequence linked to a galactose-inducible promoter between the E. coli-derived hisG sequences of about 1.2 kb recurring in the same orientation and used this construct for transformation. They inserted the construct into a chromosome and then cultured it in a medium containing galactose to successfully remove the selective marker gene at an efficiency of 96% or more. However, this method is not suitable for commercial use because the selective marker gene is excised by homologous recombination to leave an unnecessary long sequence as an excision mark of the selective gene on the chromosome.
Accordingly, it is an object of the present invention to provide a method for preparing a transformant lacking a selective marker gene and efficiently expressing a desired gene of interest. It is also an object of the present invention to apply thus prepared transformant yeast to the production of liquors or bread, and especially beer.
We found a method for preparing a yeast transformant lacking a selective marker gene by combining FRT sequences and a growth inhibition sequence in order to solve the above problems.
Specifically, the present invention provides a DNA construct comprising:
(1) a selective marker gene,
(2) a galactose-inducible growth inhibition sequence,
(3) a pair of FRT sequences in the same orientation flanking (1) and (2), and
(4) a DNA fragment capable of recombining with a yeast chromosomal DNA located at each end of (3),
wherein said FRT sequences contain the following sequence: 5xe2x80x2-GAAGTTCCTATACTTTCTAGACAATAGGACTTC-3xe2x80x2 (SEQ ID NO: 1)
inverted spacer inverted
repeat (1) sequence repeat (1)
or a sequence substantially identical to said sequence, provided that in each member of said pair of FRT sequences, the inverted repeat distal from the flanked selective marker gene and growth inhibition sequence has at least one but no more than six nucleotides deleted on the side distal from the spacer sequence.
The present invention also provides a method for transforming a yeast of the genus Saccharomyces, comprising:
(1) transferring said DNA construct into yeast cells to integrate said DNA construct into a yeast chromosome by recombination between a DNA fragment capable of recombining with a yeast chromosomal DNA present in said DNA construct and the yeast chromosomal DNA,
(2) selecting yeast cells transfected with said DNA construct based on the expression of a selective marker gene contained in said DNA construct,
(3) culturing said cells in a non-selective medium to induce recombination between a pair of FRT sequences contained in said DNA construct, thereby excising the selective marker gene, and
(4) culturing said cells in a medium containing galactose to select growable yeast cells.
The present invention also provides a yeast of the genus Saccharomyces obtained by using said transformation method.
The present invention also provides a method for producing a beer using said yeast of the genus Saccharomyces.
The present invention also provides a beer obtained by said method.